Functional characterization of a Ca(2+)-activated non-selective cation channel in human atrial cardiomyocytes

Abstract

Cardiac arrhythmias, which occur in a wide variety of conditions where intracellular calcium is increased, have been attributed to the activation of a transient inward current (Iti). Iti is the result of three different [Ca]i-sensitive currents: the Na(+)-Ca2+ exchange current, a Ca(2+)-activated chloride current and a Ca(2+)-activated non-selective cationic current. Using the cell-free configuration of the patch-clamp technique, we have characterized the properties of a Ca(2+)-activated non-selective cation channel (NSC(Ca)) in freshly dissociated human atrial cardiomyocytes. In excised inside-out patches, the channel presented a linear I-V relationship with a conductance of 19 +/- 0.4 pS. It discriminated poorly among monovalent cations (Na+ and K+) and was slightly permeable to Ca2+ ions. The channel's open probability was increased by depolarization and a rise in internal calcium, for which the Kd for [Ca2+]i was 20.8 microM. Channel activity was reduced in the presence of 0.5 mM ATP or 10 microM glibenclamide on the cytoplasmic side to 22.1 +/- 16.8 and 28.5 +/- 8.6%, respectively, of control. It was also inhibited by 0.1 mM flufenamic acid. The channel shares several properties with TRPM4b and TRPM5, two members of the 'TRP melastatin' subfamily. In conclusion, the NSC(Ca) channel is a serious candidate to support the delayed after-depolarizations observed in [Ca2+] overload and thus may be implicated in the genesis of arrhythmias.

Figure 1. Conductive properties of the non-selective cation channel

A, phase–contrast micrograph of freshly isolated human atrial cardiomyocytes. Note the shadow cast by the patch pipette used for current recordings. B, single-channel tracings recorded at various voltages from an inside-out patch. Pipette and bath contained 140 NaCl-standard solution (CaCl₂: bath 1.8 mm, pipette 1 mm). V_m corresponds to the membrane potential. Dashed lines indicate the current level of closed channels. Two similar channels are present in the patch. C, current–voltage relationship under the same ionic conditions as in B. Data points were fitted by linear regression, yielding the following values: slope conductance (g), 19 ± 0.4 pS; reversal potential (V_rev), −1.7 ± 1.3 (n = 7). D, voltage dependence of open probability (P_o). In 4 experiments similar to that shown in B, P_o was determined at various voltages using amplitude histograms. Fitting of data with a Boltzmann equation yielded the following values: V_0.5= 40 ± 10.7 mV, P_max= 0.61 ± 0.11, s = 13.5 ± 3.5 mV.

Figure 2. Channel selectivity

A, single channel current tracings recorded at various voltages from an inside-out patch. Pipette: 140 mm NaCl, bath 14 mm NaCl. B, current–voltage relationship under the same conditions as in A averaged from 7 patches. Data were fitted by the GHK equation, giving values of V_rev=+38.5 ± 4.2 mV and P_Cl/P_Na= 0.1 ± 0.04. C, current–voltage relationship in the presence of 145 mm KCl in the bath (pipette: 140 mm NaCl) (n = 6). Data points were fitted by a linear regression (g = 18.7 ± 0.1 pS; V_rev=−0.7 ± 1.7 mV, P_K/P_Na= 1.17 ± 0.08). D, current–voltage relationship averaged from 4 inside-out patches with pipette 140 mm NaCl and bath 100 mm CaCl₂. Data points were fitted by the GHK equation (g = 17.8 ± 3.5 pS; V_rev=+39.5 ± 4.1 mV, P_Ca/Na= 0.13 ± 0.02).

Figure 3. Effect of internal calcium on channel activity

A, single channel currents recorded from an inside-out patch showing that channel activity increases with rising [Ca²⁺]_i (V_m=+40 mV; pipette and bath contained 140 mm NaCl). B, corresponding amplitude histograms. ‘c’ and ‘o’ indicate the current level for the closed and open states of the channel, respectively. C, values of P_o (mean ±s.e.m.) at various [Ca²⁺]_i (V_m=+40 mV; n = 4).

Figure 4. Effect of internal ATP and glibenclamide on channel activity

A, single channel currents recorded from an inside-out patch showing the reversible inhibition of channel activity by 0.5 mm internal ATP corresponding to 16 μm free-ATP (V_m=+30 mV). Three identical channels were present in the patch membrane, labelled ‘c’ for the closed state, and 1, 2 and 3 indicating the current opening level for the three channels. Current variations at the single channel level can be seen more easily at the bottom of the panel. B, corresponding amplitude histograms for control conditions and with ATP in the bath. Current amplitude corresponding to the opening of one channel is 0.5 pA, as expected at this voltage. C, open probability of the channel as a percentage of control in the presence of 0.5 mm ATP or 10 μm glibenclamide averaged for 5 and 4 experiments, respectively. D, single channel currents recorded from an inside-out patch showing the reversible inhibition of channel activity by 10 μm glibenclamide (V_m=+40 mV)

Figure 5. Inhibition of channel activity by flufenamic acid

A, single channel currents recorded from an inside-out patch illustrating the reversible blocking effect of flufenamic acid (fl. ac., 0.1 mm) (V_m=+40 mV). ‘c’ indicates the closed channel current level while 1 and 2 indicate the current levels corresponding to the open states of one or two identical channels. B, corresponding amplitude histograms indicating a current level of 0.7 pA for the opening of one channel, as expected at this voltage. C, open probability in the absence (control) or presence of 0.1 mm flufenamic acid (V_m=+40 mV; n = 3)

Figure 6. TRPM4b mRNA expression on human atrium

A, the predicted topology of TRPM4b. The channel is composed of six membrane-spanning segments (M1 to M6) and a pore domain between M5 and M6. For PCR reactions, two pairs of primers were used. One in the segment encoding for a part of the N-terminal domain (a) and the other for a part of the pore domain (b). B, total RNA was extracted from human atrial samples and was reverse-transcribed. cDNA was amplified by PCR for 30 cycles. The two pairs of primers (a and b) were used separately and amplified products were obtained at the expected size (399 and 300 bp, respectively). ‘m’ is the 100 bp DNA size marker.